Foxp3, a 47-kDa transcription factor, is necessary and sufficient for development and function of natural Tregs. The role of Foxp3+ Tregs in controlling self-reactive T cells and preventing autoimmunity is well established. During comparison of Foxp3 expression by resting vs. activated Tregs, we detected a 41-kDa Foxp3 species in the chromatin fraction of activated Tregs that was not detectable in resting Tregs, indicating Foxp3 may be processed by proteolytic cleavage. Searches of murine and human Foxp3 sequences for potential recognition sites revealed two RXXR proprotein convertase (PC) motifs, 48RDLR51 and 414RKKR417, located near the N- terminal and C-terminal ends. In Preliminary Studies, we show Foxp3 is cleaved at both the N-terminal and C- terminal RXXR sites and N-terminal cleavage results in the generation of the 41-kDa species. Cleavage at both sites depends on the presence of intact RXXR motifs. Transduced CD4+ cells expressing Foxp3 forms mimicking N-, C-, N&C-cleaved Foxp3 are functionally distinct, as evidenced by their differing effects on expression of Treg markers, suppression of Teff cell proliferation and prevention of experimental colitis. Our results indicate the cleavage events have important functional consequences and provide a means for Foxp3 to achieve its diverse effects on gene expression. Based on our preliminary data, 2 specific aims are proposed. Aim 1: To determine the effects of Foxp3 processing on expression of critical Treg markers and to study the effect of proteolytic cleavage on the promoter binding ability of Foxp3. The qPCR analysis of CTLA-4 and IL-10 mRNA expression in CD4+ cells expressing WT Foxp3 vs. different forms of Foxp3, showed major differences between WT and processed forms of Foxp3. This analysis will be extended to other critical Treg markers. In addition, protein levels of Treg markers will be determined by ELISA, Luminex and Panomics arrays. DNA-binding of the different Foxp3 forms on promoters Foxp3 is known to directly interact with and the histone acetylation levels at these promoters will be determined by ChIP assays. Aim 2: To determine the PCs responsible in cleaving the Foxp3 at the N-terminal RDLR and C- terminal RKKR sites. In preliminary studies involving co-expression of PC1 and PC7, both PCs were capable of processing Foxp3 at the C-terminal but not at the N-terminal site. We plan to analyze all of the 7 known PCs, to determine those capable of N-terminal or C-terminal processing of Foxp3. As a second approach, shRNA technology will be used to determine the role of PCs in Foxp3 processing. Relevance to NIH and biomedical research: These studies will generate the first data on the functional roles of the different processed forms of Foxp3. Devising methods that alter the ability of the PCs to process Foxp3 should generate Tregs with functions tailored for specific therapeutic purposes. PUBLIC HEALTH RELEVANCE: Foxp3 is a DNA-binding protein that determines development and function of regulatory T cells, a group of T cells that are critical in keeping immune responses under control. Foxp3 can affect many genes, by increasing or suppressing their expression. We have shown Foxp3 is cleaved at two sites and cells expressing the cleaved forms of Foxp3 function very differently, indicating potential in therapy.